Use of acetylated tubulin as a biomarker of drug response to furazanobenzimidazoles

ABSTRACT

Use of acetylated tubulin as a biomarker for predicting the response to a compound, preferably resistance of a disease such as cancer in a subject to said compound, wherein the compound is a furazanobenzimidazoles compound of general formula (I).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/983,887, filed Sep. 16, 2013, which in turn is a National Stage ofInternational Application No. PCT/EP2012/052954, filed Feb. 21, 2012,which claims priority of European Patent Application No. 11155774.0,filed Feb. 24, 2011. The entire contents of the above-identifiedapplications are hereby incorporated by reference.

The present invention relates to use of acetylated tubulin as abiomarker for predicting the response of a disease, such as a neoplasticor autoimmune disease, preferably cancer, to a compound of generalformula I, such as3-(4-{1-[2-(4-amino-phenyl)-2-oxo-ethyl]-1H-benzoimidazol-2-yl}-furazan-3-ylamino)-propionitrile(BAL27862). In other aspects it relates to methods and kits, as well asmethods of treatment involving the use of the biomarker.

Microtubules are one of the components of the cell cytoskeleton and arecomposed of heterodimers of alpha and beta tubulin. Agents that targetmicrotubules are among the most effective cytotoxic chemotherapeuticagents having a broad spectrum of activity. Microtubule destabilisingagents (e.g. the vinca-alkaloids such as vincristine, vinblastine andvinorelbine) are used for example in the treatment of several types ofhematologic malignancies, such as lymphoblastic leukaemia and lymphoma,as well as solid tumours, such as lung cancer. Microtubule stabilisingagents (e.g. the taxanes such as paclitaxel, docetaxel) are used forexample in the treatment of solid tumours, including breast, lung andprostate cancer.

However resistance to these known microtubule targeting agents canoccur. The resistance can either be inherent or can be acquired afterexposure to these agents. Such resistance therefore impacts patientsurvival rates, as well as choices of treatment regimes. Severalpotential mechanisms of resistance have been identified, and includedefects in the microtubule targets, such as elevated levels ofbeta-tubulin subtype III and acquired mutations in beta-tubulin subtypeI that are known to reduce taxane binding. Furthermore, defects in othercell proteins have been suggested to be associated with resistance tocertain microtubule targeting agents, such as overexpression of theefflux pump P-glycoprotein (P-gp pump, also known as multi-drugresistance protein 1 or MDR1). Such factors may then be used asbiomarkers of resistance to these conventional microtubule targetingagents.

A relatively recently discovered class of microtubule destabilisingagents are compounds encompassed by the formula given below:

whereinR represents phenyl, thienyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacentsubstituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents a group C═Y, wherein Y stands for oxygen or nitrogensubstituted by hydroxy or lower alkoxy;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable salts thereof;or whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein twoadjacent substituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents oxygen;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable salts thereof;and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

These compounds are disclosed in WO2004/103994 A1, which is incorporatedby cross-reference herein. These compounds have been shown to arresttumour cell proliferation and induce apoptosis.

The synthesis of compounds of formula I is described in WO2004/103994A1, in general on pages 29-35, and specifically on pages 39-55, whichare incorporated herein by cross-reference. They may be prepared asdisclosed or by an analogous method to the processes described therein.

One compound falling within this class, known as BAL27862, and shown inWO2004/103994 A1 as example 58, and specifically incorporated byreference herein, has the structure and chemical name given below:

Chemical name:3-(4-{1-[2-(4-Amino-phenyl)-2-oxo-ethyl]-1H-benzoimidazol-2-yl}-furazan-3-ylamino)-propionitrile;or herein as Compound A

Further compounds exemplified in WO2004/103994 A1 as examples 50 and 79respectively, and also specifically incorporated by cross-referenceherein, have the structures and chemical names given below:

Chemical name:2-[2-(4-Amino-furazan-3-yl)-benzoimidazol-1-yl]-1-(4-amino-phenyl)-ethanone;or herein as Compound Band

Chemical name:3-(4-{1-[2-(6-Amino-pyridin-3-yl)-2-oxo-ethyl]-1H-benzoimidazol-2-yl}-furazan-3-ylamino)-propionitrile;or herein as Compound C.

BAL27862 has activity across a broad panel of experimental, solid tumourxenograft models. Moreover, activity is retained even against tumourmodels which were selected for resistance to conventional microtubuletargeting agents (including the vinca-alkaloid microtubule destabilisersand the microtubule stabilisers paclitaxel and epothilone B). BAL27862activity is not affected by over-expression of the P-gp pump in anymodels tested in vitro, nor in human mammary tumour xenografts.Additionally, BAL27862 retained its activity despite elevated levels ofbeta-tubulin subtype III and mutations in tubulin subtype I.

Hence, BAL27862 activity is not affected by a number of factors thatconfer resistance to conventional microtubule targeting agents.

Moreover, it is known that compounds of general formula I have adifferent effect on the phenotype of cells compared to other microtubuletargeting agents, including other microtubule destabilisers. Treatmentwith a compound of general formula I induces a consistent microtubulephenotype in tumour cell lines derived from a variety of organs, forexample lung, cervix and breast, as seen in FIGS. 1A-1F. Staining themicrotubules in these cells with an anti alpha tubulin antibody showsthat rather than the mitotic spindle fibres of untreated cells, onlydot-like structures are visible in the treated cells. This same effectis also shown using Compounds C and B in FIGS. 2A and 2B respectively onthe lung cancer cell line A549. It is however very distinct from thatobserved with the conventional microtubule targeting agents vinblastine,colchicine, paclitaxel and nocodazole as seen in FIGS. 3B, 3C, 3D and4A-4G, respectively. The microtubules were stained with an anti-alphatubulin antibody and the cells viewed at a 1000× magnification (FIGS.3A-3D, 4A-4G). For the cells treated with BAL27862, multiple dot-likestructures are visible, whereas, in stark contrast, the otherconventional drugs produce filamentous microtubule structures, or densemicrotubule aggregate structures. These differences at the phenotypiclevel, at compound doses considered optimal in terms ofantiproliferative effect, indicate a difference in the mode of action atthe molecular level.

Furthermore, it is known that BAL27862 elicits a dominant microtubulephenotype in the presence of the other microtubule targeting agents.Treatment with vinblastine, colchicine, paclitaxel or nocodazole aloneinduced the microtubule phenotypes characteristic of these agents (FIG.5A, 5D, 5G, 6C-6F respectively). However, combination treatment withBAL27862 for the last 4 hours resulted in disruption of thesephenotypes; despite the continued presence of vinblastine, colchicine,paclitaxel or nocodazole (FIG. 5B, 5E, 5H, 6G-6J respectively). Incontrast, treating first with BAL27862 and subsequently for 4 hours incombination with vinblastine, colchicine, paclitaxel or nocodazole hadno impact on generation of the phenotype consistent with BAL27862treatment (FIG. 5C, 5F, 5I, 6K-6N respectively).

These data all demonstrate that BAL27862 affects microtubule biology ina different manner than conventional microtubule targeting agents.

Thus, from information about conventional microtubule targeting agents,predictions cannot be made concerning if, or how, particular genes areinvolved in the action of compounds of formula I.

An object of the present invention is to identify factors which areassociated with response to compounds of formula I or pharmaceuticallyacceptable derivatives thereof, for example to identify factorsassociated with resistance to compounds of general formula I, inparticular BAL27862 or pharmaceutically acceptable derivatives thereof,as defined below.

It has surprisingly been found that acetylated tubulin may be used as abiomarker of response to treatment with a compound of general formula Ior pharmaceutically acceptable derivatives thereof, as defined below.

In one preferred embodiment of the invention, relatively high acetylatedtubulin levels in a tumour sample are associated with resistance toBAL27862, as described below.

Tubulin is subjected to a variety of post-translational modifications,including detyrosination/tyrosination, acetylation, glutamylation,polyglycylation, phosphorylation of serine residues and phosphorylationof tyrosine residues, making it one of the most modified proteins known.

The acetylation and deacetylation of lysines in tubulin is known tooccur, although the exact function of these changes has yet to beelucidated. The best characterised site of acetylation is on the alphatubulin lysine 40, however additional acetylation sites have beenidentified on both alpha and beta tubulin. To date two microtubuledeacetylases have been identified: histone deacetylase 6 (HDAC6) andSirtuin T2 (SirT2). Very recently alphaTAT1 and the Elp3 subunit of theElongator complex were identified as lysine 40 tubulinacetyltransferases. Furthermore, the protein known as San reportedly haslysine 252 beta-tubulin acetyltransferase activity. (A novel acetylationof beta-tubulin by San modulates microtubule polymerization via downregulating tubulin incorporation, Chih-Wen Chu et al., Mol Biol Cell.2010 Dec. 22.)

Acetylated tubulin is also known as acetylated-tubulin, acetylatedmicrotubule(s), or acetyl tubulin, and the term acetylated tubulin shallbe used herein to also encompass these synonyms. The designationacetylated tubulin, shall also encompass forms wherein otherpost-translational modifications may additionally be present. The alphaand beta tubulin which form the basis for the acetylated tubulin areknown to exist in multiple variants, subtypes and isoforms, as well asthere being multiple alpha and beta tubulin genes which give rise tothese. Preferably the tubulin which is acetylated relates to humanvariants, subtypes and isoforms of alpha and beta tubulin, morepreferably to human variants, subtypes and isoforms of alpha tubulin.Subtypes of alpha tubulin include, but are not limited to, tubulin alpha1A, tubulin alpha 1B, tubulin alpha 1C, tubulin alpha 2, tubulin alpha3C/D, tubulin alpha 4A, and tubulin alpha-8 chain isoform 1. Thesubtypes tubulin alpha 1A, tubulin alpha 1B, tubulin alpha 1C, tubulinalpha 2, tubulin alpha 3C/D and tubulin alpha 4A possess a lysine 40 andare thus a preferred subset of alpha tubulins according to theinvention. Subtypes of beta tubulin include, but are not limited to,tubulin beta chain, tubulin beta-1 chain and tubulin beta-3 chainisoform 1. The protein sequences of the alpha tubulin subtypes areaccessible via the following National Center for BiotechnologyInformation (NCBI) Reference numbers NP_006000, NP_006073, NP_116093,ABD72607, NP_005992, NP_005991 and NP_061816, and the beta tubulinsubtypes are accessible via NP_821133, NP_110400 and NP_006077respectively. In an alternatively preferred embodiment the tubulin whichis acetylated is selected from the group consisting of tubulin alpha-1C(NP_116093.1), tubulin alpha 3C/D (NP_005992.1), tubulin alpha-4A(NP_005991.1) tubulin alpha-8 chain isoform 1 (NP_061816.1), tubulinbeta chain (NP_821133.1) and tubulin beta-3 chain isoform 1(NP_006077.2). The polypeptide sequences of these are also listed inFIGS. 9-14 as SEQ ID NO. 1-6, respectively. Particularly preferably thetubulin which is acetylated is selected from the group consisting oftubulin alpha-1C, tubulin alpha 3C/D, tubulin alpha-4A and tubulinalpha-8 chain isoform 1. More particularly preferably the tubulin whichis acetylated is selected from the group consisting of tubulin alpha-1C,tubulin alpha 3C/D and tubulin alpha-4A.

One aspect of the present invention relates to use of acetylated tubulinas a biomarker for predicting the response to a compound, wherein thecompound is a compound of general formula I,

whereinR represents phenyl, thienyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacentsubstituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents a group C═Y, wherein Y stands for oxygen or nitrogensubstituted by hydroxy or lower alkoxy;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof,or whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein twoadjacent substituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents oxygen;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof;and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

Preferably the response may be of a disease in a subject. Alsopreferably the response may be to treatment, i.e. to treatment with thecompound of general formula I or pharmaceutically acceptable derivativesthereof.

The biomarker acetylated tubulin is measured ex vivo in a sample orsamples taken from the human or animal body, preferably taken from thehuman body.

In a preferred embodiment, the invention relates to use of acetylatedtubulin as a biomarker for predicting the resistance of a disease in asubject to a compound of general formula I or pharmaceuticallyacceptable derivatives thereof as defined above.

Preferably the pharmaceutically acceptable derivative is selected fromthe group consisting of a salt, solvate, pro-drug, salt of a pro-drug,polymorph and isomer of a compound of general formula I as definedabove. Pro-drugs are preferably ester and amides of naturally occurringamino acids, small peptides or pegylated hydroxy acids. More preferably,the pro-drug is an amide formed from an amino group present within the Rgroup of the compound of general formula I and the carboxy group ofglycine, alanine or lysine.

Particularly preferably the compound is

or a pharmaceutically acceptable salt thereof, preferably ahydrochloride salt, most preferably a dihydrochloride salt thereof.

Another aspect of the present invention relates to a method forpredicting the response of a disease in a subject to a compound ofgeneral formula I or pharmaceutically acceptable derivatives thereof asdefined above, comprising the steps of:

-   -   a) measuring a level of acetylated tubulin in a sample        pre-obtained from the subject to obtain a value or values        representing this level; and    -   b) comparing the value or values from step a) to a standard        value or set of standard values or to pre-treatment initiation        levels.

Further preferably the response which is to be predicted is resistance.

The measuring of a level or levels of acetylated tubulin is performed exvivo in a sample or samples pre-obtained from the subject. Pre-obtainedrefers to the fact that the sample is obtained before it is subjected toany method involving measuring the level of the biomarker, andpre-obtained is not to be understood as in relation to treatment.

In a preferred embodiment, a higher level of acetylated tubulin in thesample from the subject relative to the standard value or set ofstandard values or pre-treatment initiation levels predicts resistance.

Also preferably, the disease is a neoplastic or autoimmune disease. Morepreferably the disease is cancer. Especially preferably, the cancer isselected from the group consisting of breast cancer, prostate cancer,cervical cancer, ovarian cancer, gastric cancer, colorectal cancer (i.e.including colon cancer and rectal cancer), pancreatic cancer, livercancer, brain cancer, neuroendocrine cancer, lung cancer, kidney cancer,hematological malignancies, melanoma, T-cell leukemia, and sarcomas.More especially preferably the cancer is selected from the groupconsisting of breast cancer, cervical cancer, ovarian cancer, T-cellleukemia and lung cancer. In an especially preferred embodiment thecancer is selected from the group consisting of lung cancer, ovariancancer and T-cell leukemia.

In a further aspect, the invention relates to a method of treating aneoplastic or autoimmune disease, preferably cancer, in a subject inneed thereof, comprising measuring a level of acetylated tubulin in asample from the subject to obtain a value or values representing thislevel, and treating the subject with a compound of general formula I ora pharmaceutically acceptable derivative thereof as defined above, ifthe level of acetylated tubulin in said sample is not higher than astandard value or set of standard values or pre-treatment initiationlevels.

In yet a further aspect, the invention relates to acetylated tubulin foruse in the treatment of a neoplastic or autoimmune disease, preferablycancer, comprising measuring a level of acetylated tubulin in a samplefrom the subject to obtain a value or values representing this level,and treating the subject with a compound of general formula I or apharmaceutically acceptable derivative thereof as defined above, if thelevel of acetylated tubulin is not higher than a standard value or setof standard values or pre-treatment initiation levels.

The measuring of a level of acetylated tubulin is performed ex-vivo in asample pre-obtained from the subject.

The invention also relates in another aspect to a method of treating aneoplastic or autoimmune disease, preferably cancer, by first decreasingthe level of acetylated tubulin in a subject that has a sample with ahigher level of acetylated tubulin compared to a standard level or setof standard levels or pre-treatment initiation levels, then treating thesubject with a compound of general formula I or a pharmaceuticallyacceptable derivative thereof as defined above.

In yet another aspect the invention relates to a kit for predicting theresponse to a compound of general formula I or a pharmaceuticallyacceptable derivative thereof, as defined above, comprising reagentsnecessary for measuring a level of acetylated tubulin in a sample. Morepreferably the kit also comprises a comparator module which comprises astandard value or set of standard values to which the level ofacetylated tubulin in the sample is compared.

Furthermore preferably the kit comprises a compound of general formula Ior a pharmaceutically acceptable derivative thereof as defined above. Inan especially preferred embodiment the kit comprises a compound of thefollowing formula or a pharmaceutically acceptable salt thereof

Chemical name: S-2,6-Diamino-hexanoic acid[4-(2-{2-[4-(2-cyano-ethylamino)-furazan-3-yl]-benzoimidazol-1-yl}-acetyl)-phenyl]-amide

In a particularly preferred embodiment the pharmaceutically acceptablesalt is a dihydrochloride salt.

Another further aspect of the invention relates to a device forpredicting the response to a compound of general formula I or apharmaceutically acceptable derivative thereof as defined above,comprising reagents necessary for measuring a level of acetylatedtubulin in a sample and a comparator module comprising a standard valueor set of standard values to which the level of acetylated tubulin inthe sample is compared.

In a preferred embodiment, the reagents in the kit or device comprise acapture reagent comprising a detector for acetylated tubulin, and adetector reagent. Especially preferably the capture reagent is anantibody. Also preferably, the disease is predicted to be resistant totreatment with said compound when acetylated tubulin is higher relativeto a standard value or set of standard values or pre-treatmentinitiation levels. In a preferred embodiment, the comparator module isincluded in instructions for use of the kit. In another preferredembodiment the comparator module is in the form of a display device.

Embodiments of the present invention will now be described by way ofexample with reference to the accompanying figures. The inventionhowever is not to be understood as limited to these embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F: Show the treatment of human tumour cell lines fromdifferent histotypes with 50 nM BAL27862. The microtubules of mitotic orG2/M arrested cells were stained after 24 hours treatment with 50 nMBAL27862 or vehicle control.

FIGS. 1A and 1B: A549 NSCLC cells;

FIGS. 1C and 1D: HeLa cervical cancer cells;

FIGS. 1E and 1F: SKBR3 breast cancer cells

Vehicle control treatment: FIGS. 1A, 1C & 1E,

BAL27862 treatment: FIGS. 1B, 1D & 1F.

FIGS. 2A-2B: Show the treatment of A549 NSCLC cells with the Compounds Band C. The microtubules of mitotic or G2/M arrested A549 NSCLC cellswere stained after 24 hours treatment with 80 nM or 20 nM of Compounds Band C, respectively. The white scale bar represents 10 micrometers.

FIG. 2A: treatment with 20 nM Compound C

FIG. 2B: treatment with 80 nM Compound B

FIGS. 3A-3D: Show a comparison of treatment of cells with BAL27862compared to conventional microtubule targeting agents. Microtubules ofmitotic or G2/M arrested A549 NSCLC cells were stained after 24 hours oftreatment with 50 nM of A: BAL27862; B: vinblastine; C: colchicine; D:paclitaxel. Stacks of images taken every 1 μm were processed by usingImageJ software.

FIGS. 4A-4G: Show a comparison of treatment of A549 NSCLC cells withBAL27862 compared to nocodazole. Microtubules of mitotic or G2/Marrested cells were stained after 24 h of treatment with variousconcentrations of nocodazole (B, C & D) and BAL27862 (E, F & G). A:control, B: Nocodazole 50 nM, C: Nocodazole 100 nM, D: Nocodazole 200nM, E: BAL27862 20 nM; F: BAL27862 30 nM and G: BAL27862 50 nM. Thewhite scale bar represents 10 micrometers. Representative images of themicrotubule phenotypes observed are shown.

FIGS. 5A-5I: Show a combination of treatment with BAL27862 andconventional microtubule-targeting agents. Microtubules of mitotic orG2/M arrested A549 NSCLC cells were stained after treatment for thetimes indicated below. 50 nM BAL27862, 50 nM vinblastine, 50 nMcolchicine and 25 nM paclitaxel were used. The white scale barrepresents 10 micrometers.

FIG. 5A: 24 hours vinblastine treatment;

FIG. 5B: 24 hours vinblastine treatment with the final 4 hours includingBAL27862;

FIG. 5C: 24 hours BAL27862 treatment with the final 4 hours includingvinblastine.

FIG. 5D: 24 hours colchicine treatment;

FIG. 5E: 24 hours colchicine treatment with the final 4 hours includingBAL27862;

FIG. 5F: 24 hours BAL27862 treatment with the final 4 hours includingcolchicine.

FIG. 5G: 24 hours paclitaxel treatment;

FIG. 5H: 24 hours paclitaxel treatment with the final 4 hours includingBAL27862;

FIG. 5I: 24 hours BAL27862 treatment with the final 4 hours includingpaclitaxel.

FIGS. 6A-6N: Show a combination of treatment with BAL27862 andnocodazole. Microtubules of mitotic or G2/M arrested A549 NSCLC cellswere stained after treatment for the times indicated below. 25 nMBAL27862 and nocodazole at the concentrations indicated below were used.The white scale bar represents 10 micrometers.

FIG. 6A: 24 hours control treatment;

FIG. 6B: 24 hours of 25 nM BAL27862 treatment;

FIG. 6C: 24 hours of 50 nM nocodazole treatment

FIG. 6D: 24 hours of 100 nM nocodazole treatment

FIG. 6E: 24 hours of 150 nM nocodazole treatment

FIG. 6F: 24 hours of 200 nM nocodazole treatment

FIG. 6G: 24 hours of 50 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6H: 24 hours of 100 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6I: 24 hours of 150 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6J: 24 hours of 200 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6K: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 50 nM nocodazole;

FIG. 6L: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 100 nM nocodazole;

FIG. 6M: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 150 nM nocodazole;

FIG. 6N: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 200 nM nocodazole.

FIG. 7: Shows tumour cell lines which were selected for resistance toBAL27862 through in vitro cultivation in the presence of the compound.Based on IC₅₀ (for proliferation: A549, SKOV3, H460) or EC₅₀ (for celldeath: Jurkat) determinations, BAL27862 resistance factors versusparental lines were: A549 (3.0 fold); SKOV3 (7.6 fold—resistant 1 line);Jurkat (22.5 fold), H460 (5.3 fold) (see Table 1). Whole cell proteinextracts were prepared from parental and resistant lines and analysed byimmunoblotting for acetylated tubulin expression. Actin levels wereincluded as a loading control.

FIG. 8: Shows that increased acetylated tubulin protein levels aremaintained in SKOV3 tumour lines during resistance development. SKOV3tumour cell lines were selected for resistance to BAL27862 through invitro cultivation in the presence of BAL27862 for increasing timeperiods. Based on IC₅₀ determinations, BAL27862 resistance factorsversus parental lines were: SKOV3 resistant 1 (7.6 fold), SKOV3resistant 2 (11.6 fold) (see Table 1). Whole cell protein extracts wereprepared from parental and resistant lines and analysed byimmunoblotting for acetylated tubulin expression. Actin levels act as aloading control.

FIG. 9: Shows the protein sequence of tubulin alpha-1C chain [Homosapiens] (SEQ. ID. No. 1)

FIG. 10: Shows the protein sequence of tubulin alpha-3C/D chain [Homosapiens] (SEQ ID No. 2)

FIG. 11: Shows the protein sequence of tubulin alpha-4A chain [Homosapiens] (SEQ. ID. NO. 3)

FIG. 12: Shows the protein sequence of tubulin alpha-8 chain isoform 1[Homo sapiens] (SEQ. ID. NO. 4)

FIG. 13: Shows the protein sequence of tubulin beta chain [Homosapiens](SEQ. ID. No. 5)

FIG. 14: Shows the protein sequence of tubulin beta-3 chain isoform 1[Homo sapiens] (SEQ. ID. NO. 6)

DETAILED DESCRIPTION

Compounds of Formula I

The compounds according to the invention are represented by generalformula I:

whereinR represents phenyl, thienyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacentsubstituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents a group C═Y, wherein Y stands for oxygen or nitrogensubstituted by hydroxy or lower alkoxy;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof,or whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein twoadjacent substituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents oxygen;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof;and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

Heterocyclyl designates preferably a saturated, partially saturated orunsaturated, mono- or bicyclic ring containing 4-10 atoms comprisingone, two or three heteroatoms selected from nitrogen, oxygen and sulfur,which may, unless otherwise specified, be carbon or nitrogen linked,wherein a ring nitrogen atom may optionally be substituted by a groupselected from lower alkyl, amino-lower alkyl, aryl, aryl-lower alkyl andacyl, and a ring carbon atom may be substituted by lower alkyl,amino-lower alkyl, aryl, aryl-lower alkyl, heteroaryl, lower alkoxy,hydroxy or oxo. Examples of heterocyclyl are pyrrolidinyl, oxazolidinyl,thiazolidinyl, piperidinyl, morpholinyl, piperazinyl, dioxolanyl andtetrahydropyranyl.

Acyl designates, for example, alkylcarbonyl, cyclohexylcarbonyl,arylcarbonyl, aryl-lower alkylcarbonyl, or heteroarylcarbonyl. Loweracyl is preferably lower alkylcarbonyl, in particular propionyl oracetyl.

Preferably, the compound of general formula I according to the inventionis defined as wherein R¹ is selected from the group consisting ofhydrogen, acetyl, CH₂CH₂CN and CH₂CH₂CH₂OH.

In one preferred embodiment, the compound of general formula I accordingto the invention is selected from the group consisting of:

-   4-(1-Phenacyl-1H-benzimidazol-2-yl)-furazan-3-ylamine,-   4-[1-(4-Bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine    oxime,-   N-{4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl}-acetamide,-   4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl)-amine-   4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(3-hydroxypropyl)-amine,-   4-[1-(3-Amino-4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine-   4-[1-(3-Methoxy-4-methoxymethoxy-phenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine,    and pharmaceutically acceptable derivatives thereof.

In another preferred embodiment, the compound of general formula Iaccording to the invention is selected from the group consisting of:

whereinR, Y and R¹ are defined as follows:

R Y R¹

O H

NOH H

NOMe H

O H

NOH H

NOH H

NOMe H

O H

NOH H

NOMe H

O H

NOH H

NOMe H

O H

NOMe H

O H

O H

NOH H

NOMe H

O H

NOH H

NOMe H

NOMe H

O H

O Ac

O H

O H

O H

O CH₂CH₂CN

O CH₂CH₂CN

O H

O H

O CH₂CH₂CH₂OH

O H

O CH₂CH₂CN

O H

O CH₂CH₂CN

O CH₂CH₂CN

O CH₂CH₂CN

O H

O H

O H

O H

O H

O H

O H

O H

O CH₂CH₂CN

O H

O H

O H

O H

O H

O H

O H

O H

O H

O CH₂CH₂CN

O H

O H

O CH₂CH₂CNor pharmaceutically acceptable derivatives thereof.

In yet another preferred embodiment, the compound of general formula Iaccording to the invention is selected from the group consisting of:

-   4-(1-Phenoxymethyl-1H-benzimidazol-2-yl)-furazan-3-ylamine,-   4-[1-(4-Fluorophenoxymethyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine,-   4-[1-(3,4-Dimethylphenoxymethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl)-amine    and compounds represented by the formula:

wherein R and R¹ are as defined below

R R¹

H

H

H

H

H

CH₂CH₂CN

CH₂CH₂CN

CH₂CH₂CN

H

H

H

H

H

H

H

H

CH₂CH₂CN

CH₂CH₂CH₂OH

H

Hand pharmaceutically acceptable derivatives thereof.

In still yet another preferred embodiment the compound of generalformula I according to the invention is:

wherein R, R⁴ and R⁵ are as defined below

R R⁴ R⁵

Me Me

Me Me

Me Me

Me Me

Me Me

OMe OMe

OMe OMe

OMe OMe

OMe OMe

OMe OMeor pharmaceutically acceptable derivatives thereof.

More preferably, the compound according to the invention is a compoundof general formula I

whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from lower alkyl, lower alkoxy, amino,acetylamino, halogen and nitro;and wherein pyridinyl is optionally substituted by amino or halogen;X represents a group C═O;R¹ represents hydrogen or cyano-lower alkyl;R², R³, R⁴, R⁵ and R⁶ represent hydrogen;and pharmaceutically acceptable derivatives thereof,and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

Especially preferably, the compound according to the invention isrepresented by the following formula

wherein R, Y and R¹ are defined as follows:

R Y R¹

O H

O CH₂CH₂CN

O H

O CH₂CH₂CNor pharmaceutically acceptable derivatives thereof.

More especially preferably, the compound according to the invention isrepresented by the following formula

wherein R, Y and R1 are defined as follows:

R Y R1

O CH₂CH₂CN

O H

O CH₂CH₂CN

or pharmaceutically acceptable derivatives thereof.

Particularly preferably, the compound according to the invention is

or pharmaceutically acceptable derivatives thereof.

The term derivative or derivatives in the phrase “pharmaceuticallyacceptable derivative” or “pharmaceutically acceptable derivatives” ofcompounds of general formula I relates to salts, solvates and complexesthereof and to solvates and complexes of salts thereof, as well as topro-drugs, polymorphs, and isomers thereof (including optical, geometricand tautomeric isomers) and also salts of pro-drugs thereof. In a morepreferred embodiment, it relates to salts and pro-drugs, as well as tosalts of pro-drugs thereof.

Salts are preferably acid addition salts. Salts are formed, preferablywith organic or inorganic acids, from compounds of formula (I) with abasic nitrogen atom, especially the pharmaceutically acceptable salts.Suitable inorganic acids are, for example, halogen acids, such ashydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organicacids are, for example, carboxylic, phosphonic, sulfonic or sulfamicacids, for example acetic acid, propionic acid, octanoic acid, decanoicacid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid,succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid,malic acid, tartaric acid, citric acid, amino acids, such as glutamicacid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleicacid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoicacid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylaceticacid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid,methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid,N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamicacid, or other organic protonic acids, such as ascorbic acid.

The compound according to the invention may be administered in the formof a pro-drug which is broken down in the human or animal body to give acompound of the formula I. Examples of pro-drugs include in vivohydrolysable esters and amides of a compound of the formula I.Particular pro-drugs considered are ester and amides of naturallyoccurring amino acids and ester or amides of small peptides, inparticular small peptides consisting of up to five, preferably two orthree amino acids as well as esters and amides of pegylated hydroxyacids, preferably hydroxy acetic acid and lactic acid. Pro-drug estersare formed from the acid function of the amino acid or the C terminal ofthe peptide and suitable hydroxy group(s) in the compound of formula I.Pro-drug amides are formed from the amino function of the amino acid orthe N terminal of the peptide and suitable carboxy group(s) in thecompound of formula I, or from the acid function of the amino acid orthe C terminal of the peptide and suitable amino group(s) in thecompound of formula I. Particularly preferably the pro-drug amides areformed from the amino group(s) present within the R group of formula I.

More preferably, the pro-drug is formed by the addition of glycine,alanine or lysine to the compound of formula I.

Even more preferably the compound of general formula I is in the form ofa pro-drug selected from the compounds of formulae:

In an especially preferred embodiment the compound of general formula Iis in the form of a pro-drug which has the following formula

In a most especially preferred embodiment the compound according to theinvention is a salt, preferably a hydrochloride salt, most preferably adihydrochloride salt, of a compound of the following formula

The pharmaceutically active metabolite in vivo in this case is BAL27862.

These pro-drugs may be prepared by processes that are known per se, inparticular, a process, wherein a compound of formula (II)

wherein R¹ is defined as for formula (I) and Z is CH or N, or aderivative of such a compound comprising functional groups in protectedform,or a salt thereof is(1) acylated with an amino acid of formula (III)

whereinR¹⁰ is selected from hydrogen (Gly); methyl (Ala) and protectedaminobutyl (Lys) andR¹¹ is a suitable amino protecting group, and(2) any protecting groups in a protected derivative of the resultingcompound are removed to yield a pro-drug as shown above, and, if sodesired,(3) said pro-drug is converted into a salt by treatment with an acid, ora salt of a compound of formula (II) is converted into the correspondingfree compound of formula (II) or into another salt, and/or a mixture ofisomeric product compounds is separated into the individual isomers.

Acylation of a compound of formula (II) with an amino acid of formula(III) is performed in a manner known per se, usually in the presence ofa suitable polar or dipolar aprotic solvent, with cooling or heating asrequired, for example in a temperature range from approximately minus80° C. to approximately plus 150° C., more preferably from minus 30° C.to plus 120° C., especially in a range from approximately around 0° C.to the reflux temperature of the used solvent. Optionally a suitablebase is added, in particularly an aromatic base like pyridine orcollidine or a tertiary amine base such as triethylamine ordiisopropylethylamine, or an inorganic basic salt, e.g. potassium orsodium carbonate.

Acylation may be accomplished under conditions used for amide formationknown per se in peptide chemistry, e.g. with activating agents for thecarboxy group, such as carbodiimides likeN,N′-diethyl-,N,N′-dipropyl-,N,N′-diisopropyl-,N,N′-dicyclohexylcarbodiimideand N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide-hydrochloride(EDC), or with agents such as 1-hydroxybenzotriazole (HOBt),benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(BOP), O-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HATU),2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU), optionally in the presence of suitable bases, catalysts orco-reagents. The carboxy group may also be activated as acyl halogenide,preferably as acyl chloride, e.g. by reaction with thionylchloride oroxalylchloride, or as symmetrical or unsymmetrical anhydride, e.g. byreaction with halogeno formates like ethyl chloroformate, optionally inthe presence of suitable bases, catalysts or co-reagents.

If one or more other functional groups, for example carboxy, hydroxy oramino, are or need to be protected in a compound of formula (II) or(III), because they should not take part in the reaction, these are suchprotecting groups as are usually applied in the synthesis of amideslike, in particular peptide compounds, cephalosporins, penicillins,nucleic acid derivatives and sugars, which are known to the skilledpersons. Suitable protecting groups for amino groups are for examplet-butyl carbamate, benzyl carbamate or 9-fluorenylmethyl carbamate.

The protecting groups may already be present in precursors and shouldprotect the functional groups concerned against unwanted secondaryreactions, such as alkylations, acylations, etherifications,esterifications, oxidations, solvolysis, and similar reactions. It is acharacteristic of protecting groups that they lend themselves readily,i.e. without undesired secondary reactions, to removal, typically bysolvolysis, reduction, photolysis or also by enzyme activity, forexample under conditions analogous to physiological conditions, and thatthey are not present in the end products. The specialist knows, or caneasily establish, which protecting groups are suitable with thereactions mentioned hereinabove and hereinafter.

The protection of such functional groups by such protecting groups, theprotecting groups themselves, and their removal reactions are describedfor example in standard reference books for peptide synthesis and inspecial books on protective groups such as J. F. W. McOmie, “ProtectiveGroups in Organic Chemistry”, Plenum Press, London and New York 1973, in“Methoden der organischen Chemie” (Methods of organic chemistry),Houben-Weyl, 4th edition, Volume 15/I, Georg Thieme Verlag, Stuttgart1974, and in T. W. Greene, G. M. Wuts “Protective Groups in OrganicSynthesis”, Wiley, New York, 2006.

Disease

The compounds of general formula I according to the invention have beenshown to arrest cell proliferation and induce cell death, for example byapoptosis.

Dysregulation of cell proliferation, or lack of appropriate cell death,has wide ranging clinical implications. A number of diseases associatedwith such dysregulation involve hyperproliferation, inflammation, tissueremodeling and repair. Familiar indications in this category includecancers, restenosis, neointimal hyperplasia, angiogenesis,endometriosis, lymphoproliferative disorders, transplantation relatedpathologies (graft rejection), polyposis, loss of neural function in thecase of tissue remodeling and the like.

Cancer is associated with abnormal cell proliferation and cell deathrates. As apoptosis is inhibited or delayed in most types ofproliferative, neoplastic diseases, induction of apoptosis is an optionfor treatment of cancer, especially in cancer types which showresistance to classic chemotherapy, radiation and immunotherapy(Apoptosis and Cancer Chemotherapy, Hickman and Dive, eds., BlackwellPublishing, 1999). Also in autoimmune and transplantation relateddiseases and pathologies compounds inducing apoptosis may be used torestore normal cell death processes and therefore can eradicate thesymptoms and might cure the diseases. Further applications of compoundsinducing apoptosis may be in restenosis, i.e. accumulation of vascularsmooth muscle cells in the walls of arteries, and in persistentinfections caused by a failure to eradicate bacteria- and virus-infectedcells. Furthermore, apoptosis can be induced or reestablished inepithelial cells, in endothelial cells, in muscle cells, and in otherswhich have lost contact with extracellular matrix.

A compound according to general formula I or pharmaceutically acceptablederivatives thereof may be used for the prophylactic or especiallytherapeutic treatment of the human or animal body, in particular fortreating a neoplastic disease, autoimmune disease, transplantationrelated pathology and/or degenerative disease. Examples of suchneoplastic diseases include, but are not limited to, epithelialneoplasms, squamous cell neoplasms, basal cell neoplasms, transitionalcell papillomas and carcinomas, adenomas und adenocarcinomas, adnexaland skin appendage neoplasms, mucoepidermoid neoplasms, cysticneoplasms, mucinous and serous neoplasms, ducal-, lobular and medullaryneoplasms, acinar cell neoplasms, complex epithelial neoplasms,specialized gonadal neoplasms, paragangliomas and glomus tumors, naeviand melanomas, soft tissue tumors and sarcomas, fibromatous neoplasms,myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complexmixed and stromal neoplasms, fibroepithelial neoplasms, synovial likeneoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblasticneoplasms, mesonephromas, blood vessel tumors, lymphatic vessel tumors,osseous and chondromatous neoplasms, giant cell tumors, miscellaneousbone tumors, odontogenic tumors, gliomas, neuroepitheliomatousneoplasms, meningiomas, nerve sheath tumors, granular cell tumors andalveolar soft part sarcomas, Hodgkin's and non-Hodgkin's lymphomas,other lymphoreticular neoplasms, plasma cell tumors, mast cell tumors,immunoproliferative diseases, leukemias, miscellaneousmyeloproliferative disorders, lymphoproliferative disorders andmyelodysplastic syndromes.

The compounds of general formula I or pharmaceutically acceptablederivatives thereof may be used to treat autoimmune diseases. Examplesof such autoimmune diseases include, but are not limited to, systemic,discoid or subacute cutaneous lupus erythematosus, rheumatoid arthritis,antiphospholipid syndrome, CREST, progressive systemic sclerosis, mixedconnective tissue disease (Sharp syndrome), Reiter's syndrome, juvenilearthritis, cold agglutinin disease, essential mixed cryoglobulinemia,rheumatic fever, ankylosing spondylitis, chronic polyarthritis,myasthenia gravis, multiple sclerosis, chronic inflammatorydemyelinating polyneuropathy, Guillan-Barre syndrome,dermatomyositis/polymyositis, autoimmune hemolytic anemia,thrompocytopenic purpura, neutropenia, type I diabetes mellitus,thyroiditis (including Hashimoto's and Grave' disease), Addison'sdisease, polyglandular syndrome, pemphigus (vulgaris, foliaceus,sebaceous and vegetans), bullous and cicatricial pemphigoid, pemphigoidgestationis, epidermolysis bullosa acquisita, linear IgA disease, lichensclerosus et atrophicus, morbus Duhring, psoriasis vulgaris, guttate,generalized pustular and localized pustular psoriasis, vitiligo,alopecia areata, primary biliary cirrhosis, autoimmune hepatitis, allforms of glomerulonephritis, pulmonal hemorrhage (goodpasture syndrome),IgA nephropathy, pernicious anemia and autoimmune gastritis,inflammatory bowel diseases (including colitis ulcerosa and morbusCrohn), Behcet's disease, Celic-Sprue disease, autoimmune uveitis,autoimmune myocarditis, granulomatous orchitis, aspermatogenesis withoutorchitis, idiopatic and secondary pulmonary fibrosis, inflammatorydiseases with a possibility of autoimmune pathogensesis, such aspyoderma gangrensosum, lichen ruber, sarcoidosis (including Lofgren andcutaneous/subcutaneous type), granuloma anulare, allergic type I andtype IV immunolgical reaction, asthma bronchiale, pollinosis, atopic,contact and airborne dermatitis, large vessel vasculitis (giant cell andTakayasu's arteritis), medium sized vessel vasculitis (polyarteritisnodosa, Kawasaki disease), small vessel vasculitis (Wegener'sgranulomatosis, Churg Strauss syndrome, microscopic polangiitis,HenochSchoenlein purpura, essential cryoglobulinemic vasculitis,cutaneous leukoklastic angiitis), hypersensitivity syndromes, toxicepidermal necrolysis (Stevens-Johnson syndrome, erythema multiforme),diseases due to drug side effects, all forms of cutaneous,organ-specific and systemic effects due to type I-vu (Coombsclassification) immunologic forms of reaction, transplantation relatedpathologies, such as acute and chronic graft versus host and host versusgraft disease, involving all organs (skin, heart, kidney, bone marrow,eye, liver, spleen, lung, muscle, central and peripheral nerve system,connective tissue, bone, blood and lymphatic vessel, genito-urinarysystem, ear, cartilage, primary and secondary lymphatic system includingbone marrow, lymph node, thymus, gastrointestinal tract, includingoro-pharynx, esophageus, stomach, small intestine, colon, and rectum,including parts of above mentioned organs down to single cell level andsubstructures, e.g. stem cells).

Particularly preferably, the disease according to the invention is aneoplastic or autoimmune disease. In an especially preferred embodimentthe disease is cancer.

Examples of cancers in terms of the organs and parts of the bodyaffected include, but are not limited to, the breast, cervix, ovaries,colon, rectum, (including colon and rectum i.e. colorectal cancer),lung, (including small cell lung cancer, non-small cell lung cancer,large cell lung cancer and mesothelioma), bone, endocrine system,adrenal gland, thymus, liver, stomach, intestine, (including gastriccancer), pancreas, bone marrow, hematological malignancies, (such aslymphoma, leukemia, myeloma or lymphoid malignancies), bladder, urinarytract, kidneys, skin, thyroid, brain, head, neck, prostate and testis.Preferably the cancer is selected from the group consisting of breastcancer, prostate cancer, cervical cancer, ovarian cancer, gastriccancer, colorectal cancer, pancreatic cancer, liver cancer, braincancer, neuroendocrine cancer, lung cancer, kidney cancer, hematologicalmalignancies, melanoma, T-cell leukemia and sarcomas. More especiallypreferably the cancer is selected from the group consisting of breastcancer, cervical cancer, ovarian cancer, T-cell leukemia and lungcancer. In an especially preferred embodiment the cancer is selectedfrom the group consisting of lung cancer, ovarian cancer and T-cellleukemia.

Samples

The measurement of the level of acetylated tubulin may be performed invitro, on a sample of biological tissue derived from the subject. Thesample may be any biological material separated from the body such as,for example, normal tissue, tumour tissue, cell lines, whole blood,serum, plasma, cerebrospinal fluid, lymph fluid, circulating tumourcells, cell lysate, tissue lysate, urine and aspirates. Preferably thesample is derived from normal tissue, tumour tissue, or circulatingtumour cells. More preferably the sample is derived from tumour tissueor circulating tumour cells. In one particularly preferred embodimentthe sample is derived from tumour tissue. For example, the level ofacetylated tubulin may be measured in a fresh, frozen or formalinfixed/paraffin embedded tumour tissue sample.

The sample is pre-obtained from the subject before the sample issubjected to the method steps involving measuring the level of thebiomarker. The methods for removal of the sample are well known in theart, and it may for example be removed from the subject by biopsy, forexample by punch biopsy, core biopsy or aspiration fine needle biopsy,endoscopic biopsy, or surface biopsy. Blood may be collected byvenipuncture and further processed according to standard techniques.Circulating tumour cells may also be obtained from blood based on, forexample, size (e.g. ISET—Isolation by Size of Epithelial Tumour cells)or immunomagnetic cell enrichment. (e.g. Cellsearch®, Veridex, Raritan,N.J.).

Sample Comparison

The subject according to the invention may be human or animal.Preferably the subject is human.

The biomarker acetylated tubulin is measured ex vivo in a sample orsamples taken from the human or animal body, preferably taken from thehuman body. The sample or samples are pre-obtained from the human oranimal body, preferably pre-obtained from the human body before thesample is subjected to the method steps involving measuring the level ofthe biomarker.

A biomarker is in general a substance that is used as an indicator of abiological response, preferably as an indicator of the susceptibility toa given treatment, which in the present application is treatment with acompound of general formula I or a pharmaceutically acceptablederivative thereof.

In a particularly preferred embodiment, higher acetylated tubulin levelsin the sample relative to a standard value or set of standard valuespredicts resistance.

As used herein, an increase or relatively high or high or higher levelsrelative to a standard level or set of standard levels means the amountor concentration of the biomarker in a sample is detectably greater inthe sample relative to the standard level or set of standard levels.This encompasses at least an increase of, or higher level of, about 1%relative to the standard, preferably at least an increase of about 5%relative to the standard. More preferably it is an increase of, orhigher level of, at least about 10% relative to the standard. Moreparticularly preferably it is an increase of, or higher level of, atleast about 20% relative to the standard. For example, such an increaseof, or higher level of, may include, but is not limited to, at leastabout 1%, about 10%, about 20%, about 30%, about 50%, about 70%, about80%, about 100%, about 150% or about 200% or more relative to thestandard.

Preferably, higher acetylated tubulin levels in a sample or samples

i) relative to a standard value or set of standard values from subjectswith the same tumour histotype; or

ii) taken after treatment initiation and compared to a sample or samplestaken from the same subject before treatment initiation; or

iii) relative to a standard value or set of standard values from normalcells or tissue;

are predictive of resistance.

More preferably, higher acetylated tubulin levels in a sample or samples

i) relative to a standard value or set of standard values from subjectswith the same tumour histotype; or

ii) taken after treatment initiation and compared to a sample or samplestaken from the same subject before treatment initiation;

are predictive of resistance.

Especially preferably, higher acetylated tubulin levels in a sample orsamples taken after treatment initiation and compared to a sample orsamples taken from the same subject before treatment initiation arepredictive of resistance.

Also preferably, higher acetylated tubulin levels in a sample or samplesrelative to a standard value or set of standard values taken fromsubjects with the same tumour histotype are predictive of resistance.

In one preferred embodiment, for the case i) where the measurement iscompared in a sample or samples relative to a standard value or set ofstandard values taken from samples from subjects with the same tumourhistotype as the sample to which it is to be compared, the standardvalue or set of standard values are established from samples taken froma population of subjects with that cancer type. The samples from thesestandard subjects may for example be derived from the tumour tissue orblood, as long as the origin of the sample is consistent between thestandard and the sample to be compared.

In another preferred embodiment, for the case ii) where the measurementis compared in a sample or samples taken after treatment initiation andcompared to a sample or samples taken from the same subject beforetreatment initiation, it is measured preferably to predict acquiredresistance. The samples are compared to cells or tissue from the samebiological origin. The prediction of acquired resistance would thenindicate that the treatment with the compound should be discontinued.The biomarker is thus used to monitor whether further treatment with thecompound is likely to give the required response (e.g. reduction ofabnormal cells), or whether the cells have become non-responsive orresistant to such treatment.

In yet another preferred embodiment, for the case iii) where themeasurement is compared in a sample or samples relative to a standardvalue or set of standard values taken from normal cells or tissue, thestandard value or set of standard values may be established from asample of normal (e.g. non-tumorous) cells or tissue or body fluid. Suchdata may be gathered from a population of subjects in order to developthe standard value or set of standard values.

The standard value or set of standard values are established ex-vivofrom pre-obtained samples which may be from cell lines, or preferablybiological material taken from at least one subject and more preferablyfrom an average of subjects (e.g., n=2 to 1000 or more). The standardvalue or set of standard values may then be correlated with the responsedata of the same cell lines, or same subjects, to treatment with acompound of general formula I or a pharmaceutically acceptablederivative thereof. From this correlation a comparator module, forexample in the form of a relative scale or scoring system, optionallyincluding cut-off or threshold values, can be established whichindicates the levels of biomarker associated with a spectrum of responselevels to the compound of formula I or a pharmaceutically acceptablederivative thereof. The spectrum of response levels may compriserelative sensitivity to the therapeutic activity of the compound, (e.g.high sensitivity to low sensitivity), as well as resistance to thetherapeutic activity. In a preferred embodiment this comparator modulecomprises a cut-off value or set of values which predicts resistance totreatment.

For example, if an immunohistochemical method is used to measure thelevel of acetylated tubulin in a sample, standard values may be in theform of a scoring system. Such a system might take into account thepercentage of cells in which staining for acetylated tubulin is present.The system may also take into account the relative intensity of stainingin the individual cells. The standard values or set of standard valuesof the level of acetylated tubulin may then be correlated with dataindicating the response, especially resistance, of the subject or tissueor cell line to the therapeutic activity of a compound of formula I or apharmaceutically acceptable derivative thereof. Such data may then formpart of a comparator module.

Response is the reaction of the cell lines, or preferably of thesubject, or more preferably of the disease in a subject, to thetherapeutic activity of a compound of general formula I or apharmaceutically acceptable derivative thereof. The spectrum of responselevels may comprise relative sensitivity to the therapeutic activity ofthe compound, (e.g. high sensitivity to low sensitivity), as well asresistance to the therapeutic activity. The response data may forexample be monitored in terms of: objective response rates, time todisease progression, progression free survival, and overall survival.

The response of a cancerous disease may be evaluated by using criteriawell known to a person in the field of cancer treatment, for example butnot restricted to,

-   Response Evaluation Criteria in Solid Tumors (RECIST) Guidelines,    Source: Eisenhauer E A, Therasse P, Bogaerts J, Schwartz L H,    Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M,    Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J. New    response evaluation criteria in solid tumours: revised RECIST    guideline (version 1.1). Eur J Cancer. 2009; 45:228-47;-   RANO Criteria for High-Grade Gliomas, Source: Wen P Y, Macdonald D    R, Reardon D A, Cloughesy T F, Sorensen A G, Galanis E, Degroot J,    Wick W, Gilbert M R, Lassman A B, Tsien C, Mikkelsen T, Wong E T,    Chamberlain M C, Stupp R, Lamborn K R, Vogelbaum M A, van den Bent M    J, Chang S M. Updated response assessment criteria for high-grade    gliomas: response assessment in neuro-oncology working group. J Clin    Oncol. 2010; 28(11):1963-72;-   CA-125 Rustin Criteria for Ovarian Cancer Response, Source: Rustin G    J, Quinn M, Thigpen T, du Bois A, Pujade-Lauraine E, Jakobsen A,    Eisenhauer E, Sagae S, Greven K, Vergote I, Cervantes A,    Vermorken J. Re: New guidelines to evaluate the response to    treatment in solid tumors (ovarian cancer). J Natl Cancer Inst.    2004; 96(6):487-8;-   and-   PSA Working Group 2 Criteria for Prostate Cancer Response, Source:    Scher H I, Halabi S, Tannock I, Morris M, Sternberg C N, Carducci M    A, Eisenberger M A, Higano C, Bubley G J, Dreicer R, Petrylak D,    Kantoff P, Basch E, Kelly W K, Figg W D, Small E J, Beer T M,    Wilding G, Martin A, Hussain M; Prostate Cancer Clinical Trials    Working Group. Design and end points of clinical trials for patients    with progressive prostate cancer and castrate levels of    testosterone: recommendations of the Prostate Cancer Clinical Trials    Working Group. J Clin Oncol. 2008; 26(7): 1148-59.

Resistance is associated with there not being an observable and/ormeasurable reduction in, or absence of, one or more of the following:reduction in the number of abnormal cells, preferably cancerous cells;or absence of the abnormal cells, preferably cancerous cells; forcancerous diseases: reduction in tumour size; inhibition (i.e., slowedto some extent and preferably stopped) of further tumour growth;reduction in the levels of tumour markers such as PSA and CA-125;inhibition (i.e., slowed to some extent and preferably stopped) ofcancer cell infiltration into other organs (including the spread ofcancer into soft tissue and bone); inhibition (i.e., slowed to someextent and preferably stopped) of tumour metastasis; alleviation of oneor more of the symptoms associated with the specific cancer; and reducedmorbidity and mortality.

In a preferred embodiment resistance means there is no observable and/ormeasurable reduction in, or absence of, one or more of the followingcriteria: reduction in tumour size; inhibition of further tumour growth,inhibition of cancer cell infiltration into other organs; and inhibitionof tumour metastasis.

In a more preferred embodiment resistance refers to one or more of thefollowing criteria: no reduction in tumour size; no inhibition offurther tumour growth, no inhibition of cancer cell infiltration intoother organs; and no inhibition of tumour metastasis.

Measurement of the aforementioned resistance criteria is according toclinical guidelines well known to a person in the field of cancertreatment, such as those listed above for measuring the response of acancerous disease.

Response may also be established in vitro by assessing cellproliferation and/or cell death. For example, effects on cell death orproliferation may be assessed in vitro by one or more of the followingwell established assays: A) Nuclear staining with Hoechst 33342 dyeproviding information about nuclear morphology and DNA fragmentationwhich are hallmarks of apoptosis. B) Annexin V binding assay whichreflects the phosphatidylserine content of the outer lipid bilayer ofthe plasma membrane. This event is considered an early hallmark ofapoptosis. C) TUNEL assay (Terminal deoxynucleotidyl transferasemediated dUTP Nick End Labeling assay), a fluorescence method forevaluating cells undergoing apoptosis or necrosis by measuring DNAfragmentation by labeling the terminal end of nucleic acids. D) FACsanalysis of fluorescently labeled cells (e.g. GFP [green fluorescenceprotein]-expressing cells). Dying or dead cells show a decreasedfluorescence signal in comparison to living cells, allowing thequantification of induction of cell death. E) MTS proliferation assaymeasuring the metabolic activity of cells. Viable cells aremetabolically active whereas cells with a compromised respiratory chainshow a reduced activity in this test. F) Crystal violet staining assay,where effects on cell number are monitored through direct staining ofcellular components. G) Proliferation assay monitoring DNA synthesisthrough incorporation of bromodeoxyuridine (BrdU). Inhibitory effects ongrowth/proliferation can be directly determined. H) YO-PRO assay whichinvolves a membrane impermeable, fluorescent, monomeric cyanine, nucleicacid stain, which permits analysis of dying (e.g. apoptotic) cellswithout interfering with cell viability. Overall effects on cell numbercan also be analysed after cell permeabilisation. I) Propidium iodidestaining for cell cycle distribution which shows alterations indistribution among the different phases of the cell cycle. Cell cyclearresting points can be determined. J) Anchorage-independent growthassays, such as colony outgrowth assays which assess the ability ofsingle cell suspensions to grow into colonies in soft agar.

In a preferred embodiment relating to determination of resistance invitro, resistance means there is no decrease in the proliferation rateof abnormal cells and/or reduction in the number of abnormal cells. Morepreferably resistance means there is no decrease in the proliferationrate of cancerous cells and/or no reduction in the number of cancerouscells. The reduction in the number of abnormal, preferably cancerous,cells may occur through a variety of programmed and non-programmed celldeath mechanisms. Apoptosis, caspase-independent programmed cell deathand autophagic cell death are examples of programmed cell death. Howeverthe cell death criteria involved in embodiments of the invention are notto be taken as limited to any one cell death mechanism.

Acetylated Tubulin

Preferred examples of the protein sequence of alpha and beta tubulin(human alpha and beta tubulin) are listed in SEQ. ID No. 1-6, FIGS.9-14. Alpha or beta tubulin, especially alpha tubulin, is a precursor ofacetylated tubulin. As described previously, a specific lysine residuein the tubulin chain may be acetylated or deacetylated. The termacetylated tubulin also encompasses homologues, mutant forms, allelicvariants, isoforms, splice variants and equivalents of the sequencesrepresented by SEQ ID NO 1-6, with the proviso that a lysine residue inthe sequence is acetylated. More preferably it encompasses sequenceshaving at least about 75% identity, especially preferably at least about85% identity, particularly preferably at least about 95% identity, andmore particularly preferably about 99% identity to said sequences, within each case the proviso that a lysine residue in the sequence isacetylated. Particularly preferably lysine 40 of alpha tubulin isacetylated.

Level of Acetylated Tubulin

The level of acetylated tubulin may be assayed in the sample by proteinanalysis techniques well known to a skilled person. Examples of methodsknown in the art which are suitable to measure the level of acetylatedtubulin at the protein level include, but are not limited to, i)immunohistochemistry (IHC) analysis, ii) western blotting iii)immunoprecipitation iv) enzyme linked immunosorbant assay (ELISA) v)radioimmunoassay vi) Fluorescence activated cell sorting (FACS) vii)mass spectrometry, including matrix assisted laser desorption/ionisation(MALDI, e.g. MALDI-TOF) and electrospray ionisation mass-spectrometry(ESI-MS).

The antibodies involved in some of the above methods may be monoclonalor polyclonal antibodies, antibody fragments, and/or various types ofsynthetic antibodies, including chimeric antibodies. The antibody may belabeled to enable it to be detected or capable of detection followingreaction with one or more further species, for example using a secondaryantibody that is labeled or capable of producing a detectable result.Antibodies specific to the acetylated alpha tubulin are availablecommercially from Sigma. Additionally antibodies to acetylated alphatubulin and acetylated beta tubulin can be prepared via conventionalantibody generation methods well known to a skilled person.

Preferred methods of protein analysis are ELISA, mass spectrometrytechniques, immunohistochemistry and western blotting, more preferablywestern blotting and immunohistochemistry. In western blotting, alsoknown as immunoblotting, labelled antibodies may be used to assesslevels of protein, where the intensity of the signal from the detectablelabel corresponds to the amount of protein, and can be quantified forexample by densitometry.

Immunohistochemistry again uses labelled antibodies to detect thepresence and relative amount of the biomarker. It can be used to assessthe percentage of cells for which the biomarker is present. It can alsobe used to assess the localisation or relative amount of the biomarkerin individual cells, the latter is seen as a function of the intensityof staining.

ELISA stands for enzyme linked immunosorbant assay, since it uses anenzyme linked to an antibody or antigen for the detection of a specificprotein. ELISA is typically performed as follows (although othervariations in methodology exist): a solid substrate such as a 96 wellplate is coated with a primary antibody, which recognises the biomarker.The bound biomarker is then recognised by a secondary antibody specificfor the biomarker. This may be directly joined to an enzyme or a thirdanti-immunoglobulin antibody may be used which is joined to an enzyme. Asubstrate is added and the enzyme catalyses a reaction, yielding aspecific colour. By measuring the optical density of this colour, thepresence and amount of the biomarker can be determined.

Uses of Biomarker

The biomarker may be used to predict acquired resistance of the diseasein a subject to the compound of general formula I or a pharmaceuticallyacceptable derivative thereof as defined above.

The biomarker may be used to select subjects suffering or predisposed tosuffering from a disease, preferably cancer, for treatment with acompound of general formula I or a pharmaceutically acceptablederivative thereof as defined above. The levels of such a biomarker maybe used to identify subjects likely to respond or to not respond or tocontinue to respond or to not continue to respond to treatment with suchagents. Preferably the levels of such a biomarker may be used toidentify subjects likely to continue to respond or to not continue torespond to treatment with such agents. Stratification of subjects may bemade in order to avoid unnecessary treatment regimes. In particular thebiomarker may be used to identify subjects from whom a sample or samplesdo not display a higher level of acetylated tubulin, relative to astandard level or set of standard levels, whereupon such subjects maythen be selected for treatment with the compound of formula I or apharmaceutically acceptable derivative thereof as defined above. Moreparticularly the biomarker may be used to identify subjects from whom asample or samples do not display a higher level of acetylated tubulin,relative to a level measured before treatment initiation, whereupon suchsubjects may then be selected for continued treatment with the compoundof formula I or a pharmaceutically acceptable derivative thereof asdefined above.

The biomarker may also be used to assist in the determination oftreatment regimes, regarding amounts and schedules of dosing.Additionally, the biomarker may be used to assist in the selection of acombination of drugs to be given to a subject, including a compound orcompounds of general formula I or a pharmaceutically acceptablederivative thereof, and another chemotherapeutic (cytotoxic) agent oragents. Furthermore, the biomarker may be used to assist in thedetermination of therapy strategies in a subject including whether acompound of general formula I or a pharmaceutically acceptablederivative thereof is to be administered in combination with targetedtherapy, endocrine therapy, radiotherapy, immunotherapy or surgicalintervention, or a combination of these.

Acetylated tubulin may also be used in combination with other biomarkersto predict the response to a compound of general formula I or apharmaceutically acceptable derivative thereof and to determinetreatment regimes. It may furthermore be used in combination withchemo-sensitivity testing to predict resistance and to determinetreatment regimes. Chemo-sensitivity testing involves directly applyinga compound of general formula I to cells taken from the subject, forexample from a subject with haematological malignancies or accessiblesolid tumours, for example breast, head and neck cancers or melanomas,to determine the response of the cells to the compound.

Method of Treatment

The invention also involves in some aspects a method of treatment andacetylated tubulin for use in a method of treatment, wherein the levelof acetylated tubulin is first established relative to a standard levelor set of standard levels or pre-treatment initiation levels and then acompound of general formula I or a pharmaceutically acceptablederivative thereof as defined above, is administered if the level ofacetylated tubulin in said sample is not higher than standard value orset of standard values or has not increased relative to pre-treatmentinitiation levels respectively. The compound of formula I or apharmaceutically acceptable derivative thereof may be administered in apharmaceutical composition, as is well known to a person skilled in theart. Suitable compositions and dosages are for example disclosed in WO2004/103994 A1 pages 35-39, which are specifically incorporated byreference herein. Compositions for enteral administration, such asnasal, buccal, rectal or, especially, oral administration, and forparenteral administration, such as intravenous, intramuscular orsubcutaneous administration, to warm-blooded animals, especially humans,are especially preferred. More particularly, compositions forintravenous administration are preferred.

The compositions comprise the active ingredient and a pharmaceuticallyacceptable carrier. An example of a composition includes, but is notlimited to, the following: 5000 soft gelatin capsules, each comprisingas active ingredient 0.05 g of one of the compounds of general formula(I), are prepared as follows: 250 g pulverized active ingredient issuspended in 2 liter Lauroglykol® (propylene glycol laurate, GattefosséS. A., Saint Priest, France) and ground in a wet pulverizer to produce aparticle size of about 1 to 3 μm. 0.419 g portions of the mixture arethen introduced into soft gelatin capsules using a capsule-fillingmachine.

The invention also relates in one aspect to a method of treating aneoplastic or autoimmune disease, preferably cancer, by first decreasingthe level of acetylated tubulin in a subject that has a sample with ahigher level of acetylated tubulin compared to a standard level or setof standard levels or pre-treatment initiation levels, and then treatingthe subject with a compound of general formula I or a pharmaceuticallyacceptable derivative as defined above. The level of acetylated tubulinmay be decreased by direct or indirect chemical or genetic means.Examples of such methods are treatment with a drug that results inreduced acetylated tubulin expression, targeted delivery of viral,plasmid or peptide constructs, or antibody or siRNA or antisense todownregulate the level of acetylated tubulin. For example siRNA may beused to reduce the level of alphaTAT1 or delivery of a plasmid may beused to increase the expression of SirT2, and thereby reduce the levelof acetylated tubulin in the cell. The subject may then be treated witha compound of general formula I or a pharmaceutically acceptablederivative thereof.

A compound of general formula I or a pharmaceutically acceptablederivative thereof can be administered alone or in combination with oneor more other therapeutic agents. Possible combination therapy may takethe form of fixed combinations, or the administration of a compound ofthe invention and one or more other therapeutic agents which arestaggered or given independently of one another, or the combinedadministration of fixed combinations and one or more other therapeuticagents. A compound of general formula I or a pharmaceutically acceptablederivative thereof can, besides or in addition, be administeredespecially for tumour therapy in combination with chemotherapy(cytotoxic therapy), targeted therapy, endocrine therapy, radiotherapy,immunotherapy, surgical intervention, or a combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Otherpossible treatments are therapy to maintain the patient's status aftertumour regression, or even chemo-preventive therapy, for example inpatients at risk.

Kit and Device

In one aspect the invention relates to a kit and in another aspect to adevice for predicting the response, preferably of a disease in asubject, to a compound of general formula I or a pharmaceuticallyacceptable derivative thereof as defined above, comprising reagentsnecessary for measuring the level of acetylated tubulin in a sample.Preferably, the reagents comprise a capture reagent comprising adetector for acetylated tubulin and a detector reagent.

The kit and device may also preferably comprise a comparator modulewhich comprises a standard value or set of standard values to which thelevel of acetylated tubulin in the sample is compared. In a preferredembodiment, the comparator module is included in instructions for use ofthe kit. In another preferred embodiment the comparator module is in theform of a display device, for example a strip of colour or numericallycoded material which is designed to be placed next to the readout of thesample measurement to indicate resistance levels. The standard value orset of standard values may be determined as described above.

The reagents are preferably antibodies or antibody fragments whichselectively bind to acetylated tubulin. These may for example be in theform of one specific primary antibody which binds to acetylated tubulinand a secondary antibody which binds to the primary antibody, and whichis itself labelled for detection. Alternatively, the primary antibodymay also be labelled for direct detection. The kits or devices mayoptionally also contain a wash solution(s) that selectively allowsretention of the bound biomarker to the capture reagent as compared withother biomarkers after washing. Such kits can then be used in ELISA,western blotting, flow cytometry, immunohistochemistry or otherimmunochemical methods to detect the level of the biomarker.

More preferably the kit comprises a compound of general formula I, or apharmaceutically acceptable derivative thereof as defined above. Thiscompound may then be administered to the subject, in accordance with thelevel of the biomarker in the sample from the subject, as measured bythe reagents comprised in the kit. Therefore the kit according to theinvention may be used in the method of treatment according to theinvention, as defined above. In an especially preferred embodiment thekit comprises a compound of the following formula or a pharmaceuticallyacceptable salt thereof

In a particularly preferred embodiment of the kit the pharmaceuticallyacceptable salt is a dihydrochloride salt. In another aspect theinvention relates to the use of such a kit as described above.

Furthermore the device may comprise imaging devices or measurementdevices (for example, but not restricted to, measurement offluorescence) which further process the measured signals and transferthem into a scale in a comparator module.

In the present specification the words “comprise” or “comprises” or“comprising” are to be understood as to imply the inclusion of a stateditem or group of items, but not the exclusion of any other item or groupof items.

Experimental Methodology

Immunofluorescent Staining of Cultured Cells

A549 human non-small cell lung cancer (NSCLC, ATCC reference numberCCL-185) cells, HeLa cervical cancer cells (ATCC reference number CCL-2)and SKBR3 breast carcinoma cells (ATCC reference number HTB-30) wereseeded at densities of 50% on round microscope coverslips and culturedfor 24 hours in RPMI-1640 containing 10% FCS (also referred to as FBS)at 37° C., 5% CO₂. Compounds to be tested were dissolved in DMSO. Thecell culture medium was replaced with medium containing the dilutedcompound(s) (paclitaxel, vinblastine, colchicine and nocodazole werepurchased from Sigma-Aldrich) or vehicle. After treatment for the timesindicated in the Brief Description of the Figures, coverslips werewashed and cells were fixed in methanol/acetone (1:1) for 5 minutes atroom temperature and subsequently incubated in blocking buffer (0.5% BSAand 0.1% TX-100 in PBS) for 30 minutes at room temperature. Specimenswere then incubated with anti-alpha tubulin antibody (Sigma, 1:2000) for1 hour at room temperature in blocking buffer. After several washingsteps cells were incubated with AlexaFluor-488 goat-anti-mouse IgG(Molecular Probes, 1:3000) for 1 hour at room temperature followed byseveral washing steps with blocking buffer. Specimens were then mountedwith ProLong Gold antifade (Molecular Probes) sealed with nail polishand examined with a Leica immunofluorescence microscope. Images werecaptured with a cooled CCD-camera and processed by ImageJ software.

Generation and Crystal Violet or Cell Death Assay of BAL27862-ResistantCell Lines

BAL27862-resistant sublines of human non-small cell lung cancer (H460ATCC reference HTB-177; A549 ATCC reference CCL-185), ovarian cancer(SKOV3 ATCC reference HTB-77) and T-cell leukemia (Jurkat ATCC referenceTIB-152) cell lines were generated by long-term selection in completecell culture medium (RPMI-1640 containing 10% FBS; Sigma-Aldrich) byincreasing BAL27862 concentrations in a stepwise fashion. Dependent onthe cell line, the selection process was carried out for 8-12 months inorder to achieve resistance factors (ratio of IC₅₀ or EC₅₀ of resistantcell line versus the IC₅₀ or EC₅₀ of the appropriate parental cell line)between 3 and 22.5. The resistance factors were determined for theadherent cell lines H460, A549 and SKOV3 by measuring proliferationusing the Crystal Violet assay, whereas for the suspension Jurkat cellline cell death was measured using FACS. The resistant sub lines wereexpanded at the highest tolerated BAL27862 concentration andsubsequently frozen and stored in liquid nitrogen.

A549 (2000 cells/well), H460 (1000 cells/well) and SKOV3 (2000cells/well) cells were seeded in 96 well plates. After 24 hoursincubation, the cells were incubated for 72 hours with DMSO, BAL27862,colchicine, nocodazole, paclitaxel or vinblastine diluted in completemedium (final concentration DMSO max. 0.5%). After medium was removed,cells were fixed and stained by adding 50 μl Crystal Violet stain (0.2%Crystal Violet in 50% Methanol) per well. Plates were incubated for 1hour at room temperature. Subsequently the stain was decanted and plateswere washed 4 times with double-distilled water. Plates were air-driedfor several hours. Stain was dissolved by adding 100 μl buffer (0.1 MTris pH 7.5, 0.2% SDS, 20% Ethanol) per well and shaking the plates.Absorbance at 590 nm was measured using a SpectraMax M2e plate reader(Molecular Devices).

Jurkat cells stably expressing GFP (green fluorescence protein) wereincubated with increasing BAL27862 concentrations in culture medium for72 hours and then analyzed by FACS (excitation at 488 nM, emission at525 nM). Dying or dead cells, showing a decreased fluorescence activityin comparison to living cells, were quantified (Green fluorescentprotein as a novel tool to measure apoptosis and necrosis. Strebel etal, 2001, Cytometry 43(2): 126-133).

Antiproliferative IC₅₀ and cell death EC₅₀ values were calculated fromconcentration response curves using GraphPad Prism software. Resistancefactors were calculated as a ratio of the IC₅₀ or EC₅₀ in the resistantline variant versus the IC₅₀ or EC₅₀ in the parental line.

Protein Analysis

Cells were washed with ice-cold PBS containing 1 mM phenylmethylsulfonylfluoride (PMSF) and with ice-cold buffer containing 50 mM HEPES (pH7.5), 150 mM NaCl, 25 mM β-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mMEDTA, 15 mM pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodiummolybdate, leupeptin (10 μg/mL), aprotinin (10 μg/mL) and 1 mM PMSF.Cells were extracted in the same buffer containing 1% NP40. Afterhomogenization, lysates were clarified by centrifugation and frozen at−80° C.

Protein concentration was determined with the BCA Protein Assay(Pierce). Immunoblotting was performed using 20 μg of total protein perlane. Protein was separated on a 10% SDS-gel and transferred to a PVDFmembrane using Semidry Blotting (90 min, 50 mA/gel). Tubulin acetylationwas measured using a mouse monoclonal anti-acetylated tubulin antibody(Sigma, reference number T7451): 1:10,000 dilution in PBS 5% milk/0.1%Tween. The actin antibody used was a mouse monoclonal (Chemicon,reference number MAB1501): 1:5,000 dilution in PBS 5% milk/0.1% Tween.The secondary antibody used for immunoblotting was peroxidase-conjugatedgoat anti-mouse (Jackson ImmunoResearch Laboratories INC: #115-035-146JIR), used at a dilution of 1:5,000 in PBS plus 0.5% milk and 0.1%Tween. Labelled bands were revealed using a Raytest Stella 3200 HighPerformance Imaging System.

DETAILED EXAMPLES Example 1: A Distinct Mitotic Phenotype Induced byCompounds of General Formula I

Treatment with compound A (BAL27862) or with compound B or compound C,induced a highly reproducible and distinct microtubule phenotype in alltumour cell lines tested (shown for compound A in A549, HeLa and SKBR3cells in FIGS. 1A-1F, and for compound B and compound C in A549 cells inFIGS. 2A-2B). In dividing cells an apparent fragmentation of the mitoticspindle occurred, resulting in the formation of dot-like structures(FIGS. 1A-1F). This phenotype was shown to be distinct from thatobserved with conventional microtubule targeting agents, such as themicrotubule stabiliser paclitaxel and the microtubule destabilisersvinblastine and colchicine (FIGS. 3A-3D) and nocodazole (FIGS. 4A-4G).

Example 2: BAL27862 Overcomes Microtubule Phenotype Induced byConventional Microtubule-Targeting Drugs in a Dominant Fashion

In order to show the uniqueness of its activity on microtubules,BAL27862 was tested in combination with vinblastine, colchicine andpaclitaxel (FIGS. 5A-5I) and nocodazole (FIGS. 6A-6N) using A549 cells.Treatment with vinblastine, colchicine, paclitaxel or nocodazole aloneinduced the mitotic microtubule phenotypes characteristic of theseagents. However, combination treatment with BAL27862 for the last 4hours resulted in disruption of the microtubule structures; creating aphenotype consistent with treatment of BAL27862 alone, despite thecontinued presence of vinblastine, colchicine, paclitaxel or nocodazole.In contrast, treating first with BAL27862 and subsequently for 4 hoursin combination with vinblastine, colchicine, paclitaxel or nocodazolehad no impact on the observed microtubule phenotype that was consistentwith treatment with BAL27862.

These data demonstrate that compounds of formula I affect microtubulebiology consistently, but in a different manner than conventionalmicrotubule targeting agents.

DETAILED EXAMPLES ACCORDING TO THE INVENTION Example 3: IncreasedAcetylated Tubulin Expression is Observed in Tumour Lines Selected forResistance to a Compound of General Formula I

In vitro selection for resistance to BAL27862 resulted in the generationof 4 relatively resistant tumour cell lines, with the followingresistance factors versus parental lines (based on IC₅₀ [A549, SKOV3,H460] or EC₅₀ [Jurkat] determinations using the Crystal Violet or FACscell death assay, respectively): A549 (3.0 fold); SKOV3 resistant 1 (7.6fold); SKOV3 resistant 2 (11.6 fold); H460 (5.3 fold); Jurkat (22.5Fold) (Table 1).

TABLE 1 Resistance factors (ratio IC₅₀ or EC₅₀ BAL27862-resistant cellline variant versus IC₅₀ or EC₅₀ parental cell line) Treatment SKOV3SKOV3 compound A549 H460 resistant 1 resistant 2 Jurkat BAL27862 3.0 5.37.6 11.6 22.5 Colchicine 0.9 1.6 2.0 2.8 nd Nocodazole 1.6 1.3 3.6 3.9nd Vinblastine 2.3 4.6 15.7 17.8 nd Paclitaxel 0.06 0.3 0.4 0.5 nd nd:not determined

In general these BAL27862-resistant cells exhibited a different level ofresponse to other microtubule destabilising agents, such as colchicine,nocodazole and vinblastine, as compared to BAL27862; and indeedincreased sensitivity to the microtubule stabiliser paclitaxel wasobserved in all lines tested (Table 1).

Extraction and immunoblot analysis of these lines to measure theacetylated tubulin levels, followed by comparison to BAL27862 resistancedata (Table 1), showed that acetylated tubulin is higher in theresistant lines, as compared to the parental lines (FIG. 7). This wasmaintained throughout resistance development in the SKOV3 cells (FIG.8). These data show the association of increased acetylated tubulinexpression levels with acquired resistance to BAL27862.

LIST OF ABBREVIATIONS

-   A549 human non-small cell lung cancer cell line-   alphaTAT1 alpha-tubulin N-acetyltransferase 1-   BCA bicinchoninic acid-   BrdU bromodeoxyuridine-   BSA bovine serum albumin-   CA-125 cancer antigen 125-   CCD charge-coupled device-   cDNA complementary deoxyribonucleic acid-   CREST limited scleroderma syndrome-   DMSO dimethylsulphoxide-   DNA deoxyribonucleic acid-   dUTP 2′-Deoxyuridine 5′-Triphosphate-   EDTA Ethylenediaminetetraacetic acid-   EGTA Ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid-   ELISA enzyme-linked immunosorbent assay-   Elp3 elongator complex protein 3-   ESI-MS electrospray ionisation mass-spectrometry-   FACS fluorescence activated cell scan/sorting-   FCS/FBS foetal calf/foetal bovine serum-   G2/M transition from G2 to the mitotic phase in the cell cycle-   GFP green fluorescence protein-   HDAC6 histone deacetylase 6-   HeLa human squamous cell cancer cell line-   HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulphonic acid-   IHC Immunohistochemistry-   ISET Isolation by size of epithelial tumor cells-   Jurkat human T-cell leukemia cell line-   MAB monoclonal antibody-   MALDI matrix-assisted-laser-desorption/ionisation mass-spectrometry-   MALDI-TOF    matrix-assisted-laser-desorption/ionisation-time-of-flight-mass-spectrometry-   MDR1 multi-drug resistant protein-   mRNA messenger ribonucleic acid-   MTS    3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium-   NCBI National center for Biotechnology Information-   NSCLC non-small cell lung cancer-   NP40 Nonidet P40-   PBS phosphate buffered saline-   P-gp P-glycoprotein-   PMSF phenylmethylsulphonyl fluoride-   PSA prostate-specific antigen-   PVDF polyvinylidene fluoride-   RANO response assessment for high-grade gliomas-   RECIST response evaluation criteria in solid tumors-   RPMI-1640 cell culture medium used for culturing transformed and    non-transformed eukaryotic cells and cell lines-   SDS sodium dodecyl sulphate-   SEQ. ID No. sequence identification number-   siRNA small inhibitory ribonucleic acid-   SirT2 NAD-dependent deacetylase sirtuin-2-   SKBR3 human mammary carcinoma cell line-   SKOV3 human ovarian carcinoma cell line-   TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling-   Tween polysorbate 20, non-ionic detergent-   TX-100 Triton-X100-   YO-PRO fluorescent, monomeric cyanine, nucleic acid stain

The invention claimed is:
 1. A method of treating a neoplastic diseasein a patient in need thereof, said method comprising the steps of: a)determining the level of the acetylated tubulin proteins in a sample ofbiologic material obtained from the body of said patient; and b)administering to said patient a compound of general formula I

wherein R presents phenyl, thienyl or pyridinyl wherein phenyl isoptionally substituted by one or two substituents independently selectedfrom alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-loweralkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-loweralkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, loweralkylcarbonyloxy, amino, monoalkylamino, dialkylamino, loweralkoxycarbonylamino, lower alkylcarbonylamino, substituted amino whereinthe two substituents on nitrogen form together with the nitrogenheterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano,halogen, and nitro; and wherein two adjacent substituents aremethylenedioxy; and wherein pyridinyl is optionally substituted by loweralkoxy, amino or halogen; X represents a group C═Y, wherein Y is oxygenor nitrogen substituted by hydroxy or lower alkoxy; or when R1 is phenylor pyridinyl, X is additionally oxygen; R¹ represents hydrogen, loweralkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R², R³ and R⁶represent hydrogen; R⁴ and R⁵, independently of each other, representhydrogen, lower alkyl or lower alkoxy; or R⁴ and R⁵ together representmethylenedioxy; or pharmaceutically acceptable derivatives thereof, andwherein the term lower denotes a radical having up to 7 carbon atoms, ifthe level of acetylated tubulin proteins in said sample is not higherthan a standard value or set of standard values for the level ofacetylated tubulin proteins; wherein the neoplastic disease selectedfrom the group consisting of epithelial neoplasms, squamous cellneoplasms, basal cell neoplasms, transitional cell papillomas andcarcinomas, adenomas and adenocarcinomas, adnexal and skin appendageneoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous andserous neoplasms, ducal-, lobular and medullary neoplasms, acinar cellneoplasms, complex epithelial neoplasms, specialized gonadal neoplasms,paragangliomas and glomus tumours, naevi and melanomas, soft tissuetumours and sarcomas, fibromatous neoplasms, myxomatous neoplasms,lipomatous neoplasms, myomatous neoplasms, complex mixed and stromalneoplasms, fibroepithelial neoplasms, synovial like neoplasms,mesothelial neoplasms, germ cell neoplasms, trophoblastic neoplasms,mesonephromas, blood vessel tumours, lymphatic vessel tumours, osseousand chondromatous neoplasms, giant cell tumours, miscellaneous bonetumours, odontogenic tumours, gliomas, neuroepitheliomatous neoplasms,meningiomas, nerve sheath tumours, granular cell tumours and alveolarsoft part sarcomas, Hodgkin's and non-Hodgkin's lymphomas, otherlymphoreticular neoplasms, plasma cell tumours, mast cell tumours,immunoproliferative diseases, leukemias, miscellaneousmyeloproliferative disorders, lymphoproliferative disorders andmyelodysplastic syndromes.
 2. The method of claim 1, wherein saidpatient is an animal or human being and the level of acetylated tubulinproteins is measured ex vivo in the sample taken from said animal orhuman being.
 3. The method according to claim 2, wherein the sample isderived from normal tissue, tumor tissue, circulating tumor cells,plasma or whole blood.
 4. The method according to claim 2, wherein thesample is derived from tumor tissue or circulating tumor cells.
 5. Themethod of claim 3, wherein a higher level of acetylated tubulin proteinsin the sample relative to a standard value or a set of standard valuespredicts resistance to treating said disease with said compound offormula I or pharmaceutically acceptable derivatives thereof.
 6. Themethod of claim 5, wherein the determination of a higher level ofacetylated tubulin in said sample obtained from the animal or humanbeing is carried out by comparing the measured acetylated tubulinprotein level in said sample i) relative to a standard value or a set ofstandard values of levels of acetylated tubulin proteins from samplesfrom other subjects having the same tumour histotype as said animal orhuman being; or ii) taken after treatment initiation and compared to asample or samples taken from the same subject before treatmentinitiation; or iii) relative to a standard value or a set of standardvalues of levels of acetylated tubulin proteins from a sample or samplesof levels of acetylated tubulin from normal tissue.
 7. The method ofclaim 6, wherein the acetylated tubulin proteins are human alpha or betatubulin, wherein a lysine residue in the protein sequence of the alphaor beta tubulin is acetylated.
 8. The method of claim 7, wherein theprotein sequence of acetylated tubulin is selected from the groupconsisting of SEQ ID No. 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQID 6, and homologues, mutant forms, allelic variants, isoforms, splicevariants and proteins with sequences having at least 75% identity to SEQID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, or SEQ ID 6, with theproviso that a lysine residue in the protein sequence of the alpha orbeta tubulin is acetylated.
 9. The method of claim 6, wherein theacetylated tubulin proteins are human alpha tubulin proteins whichcomprise a lysine residue in position 40 of the protein sequence of analpha tubulin protein, which is acetylated.
 10. The method of claim 9,wherein the protein sequence of the acetylated tubulin proteins areselected from the group consisting of SEQ ID No. 1, SEQ ID 2, SEQ ID 3,and homologues, mutant forms, allelic variants, isoforms, splicevariants and proteins with sequences having at least 75% identity to SEQID 1, SEQ ID 2 or SEQ ID 3 comprising a lysine residue in position 40 ofthe sequence and wherein said lysine residue in position 40 isacetylated.
 11. The method of claim 1, wherein the compound is acompound of general formula I wherein R represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from lower alkyl, lower alkoxy, amino,acetylamino, halogen and nitro; and wherein pyridinyl is optionallysubstituted by amino or halogen; X represents a group C═O; R¹ representshydrogen or cyano-lower alkyl; R², R³, R⁴, R⁵ and R⁶ represent hydrogen;or pharmaceutically acceptable derivatives thereof, and wherein the termlower denotes a radical having up to and including a maximum of
 7. 12.The method according to claim 11, wherein said patient is an animal orhuman being and the level of acetylated tubulin proteins is measured exvivo in a sample taken from the animal or human being's body.
 13. Themethod according to claim 12, wherein the sample is derived from normaltissue, tumor tissue, circulating tumor cells, plasma or whole blood.14. The method according to claim 12, wherein the sample is derived fromtumor tissue or circulating tumor cells.
 15. The method of claim 13,wherein a higher level of acetylated tubulin proteins in a sampleobtained from said animal or human being relative to a standard value ora set of standard values predicts resistance to a therapy with compoundsof formula I or pharmaceutically acceptable derivatives thereof.
 16. Themethod of claim 15, wherein the determination of a higher level ofacetylated tubulin in said sample obtained from the animal or humanbeing is carried out by comparing the measured acetylated tubulinprotein level in said sample i) relative to a standard value or a set ofstandard values of levels of acetylated tubulin proteins from samplesfrom other subjects having the same tumour histotype as said animal orhuman being; or ii) taken after treatment initiation and compared to asample or samples taken from the same subject before treatmentinitiation; or iii) relative to a standard value or a set of standardvalues of levels of acetylated tubulin proteins from a sample or samplesof levels of acetylated tubulin from normal tissue.
 17. The method ofclaim 1, wherein the acetylated tubulin proteins are a biomarker toselect subjects suffering or predisposed to suffering from theneoplastic disease for treatment with a compound of general formula I orpharmaceutically acceptable derivatives thereof.
 18. The method of claim11, wherein the acetylated tubulin proteins are a biomarker to selectsubjects suffering or predisposed to suffering from the neoplasticdisease for treatment with a compound of general formula I orpharmaceutically acceptable derivatives thereof.
 19. The method of claim11, wherein the compound is represented by the following formula

wherein R, Y and R1 are defined as follows: R Y R¹

O CH₂CH₂CN

O H

O CH₂CH₂CN

or pharmaceutically acceptable derivatives thereof.
 20. The method ofclaim 11, wherein the compound is

or pharmaceutically acceptable derivatives thereof.
 21. The method ofclaim 11, wherein a pharmaceutically acceptable derivative is selectedfrom the group consisting of a salt, solvate, pro-drug, salt of apro-drug, polymorph and isomer of the compound of general formula I. 22.The method of claim 21, wherein the pharmaceutically acceptable pro-drugis an amide formed from an amino group present within the R group of thecompound of formula I as defined in claim 12 and the carboxy group ofglycine, alanine or lysine.
 23. The method of claim 11 wherein apharmaceutically acceptable derivative is selected from the groupconsisting of a salt, solvate, pro-drug, salt of a pro-drug, polymorphand isomer of the compound of general formula I as defined in claim 20.24. The method of claim 23, wherein the pharmaceutically acceptablepro-drug is an amide formed from an amino group present within the Rgroup of the compound of formula I as defined in claim 12 and thecarboxy group of glycine, alanine or lysine.
 25. The method of claim 11,wherein the compound is

or a pharmaceutically acceptable salt thereof.
 26. The method of claim1, wherein the neoplastic disease is selected from the group consistingof breast cancer, prostate cancer, cervical cancer, ovarian cancer,gastric cancer, colorectal cancer, pancreatic cancer, liver cancer,brain cancer, neuroendocrine cancer, lung cancer, kidney cancer,hematological malignancies, melanoma, T-cell leukemia, and sarcomas. 27.The method of claim 1, wherein the neoplastic disease is selected fromthe group consisting of breast cancer, cervical cancer, ovarian cancer,T-cell leukemia and lung cancer.
 28. The method of claim 1, wherein theneoplastic disease is selected from the group consisting of lung cancer,ovarian cancer and T-cell leukemia.
 29. The method of claim 1, whereinthe neoplastic disease is selected from the group consisting of breast,cervix, ovaries, colon, rectum (including colon and rectum i.e.colorectal cancer) lung (including small cell lung cancer, non-smallcell lung cancer, large cell lung cancer and mesothelioma), endocrinesystems, bone, adrenal gland, thymus, liver, stomach, intestine,(including gastric cancer), pancreas, bone marrow, hematologicalmalignancies (such as lymphoma, leukemia, myeloma or lymphoidmalignancies), bladder, urinary tract, kidneys, skin, thyroid, brain,head, neck, prostate and testis.
 30. The method of claim 20, wherein thedisease is breast cancer.
 31. The method of claim 20, wherein thedisease is ovarian cancer.
 32. The method of claim 20, wherein thedisease is colorectal cancer.
 33. The method of claim 20, wherein thedisease is lung cancer.
 34. The method of claim 20, wherein the diseaseis liver cancer.
 35. The method of claim 20, wherein the disease isgastric cancer.
 36. The method of claim 20, wherein the disease ispancreatic cancer.
 37. The method of claim 20, wherein the disease ishematological malignancy.
 38. The method of claim 20, wherein thedisease is kidney cancer.
 39. The method of claim 20, wherein thedisease is skin cancer.
 40. The method of claim 20, wherein the diseaseis brain cancer.
 41. The method of claim 20, wherein the disease isprostate cancer.
 42. The method of claim 20, wherein the disease is headand neck cancer.
 43. The method of claim 20, wherein the disease is asarcomas.
 44. The method of claim 20, wherein the disease is glioma. 45.The method of claim 25, wherein the disease is breast cancer.
 46. Themethod of claim 25, wherein the disease is ovarian cancer.
 47. Themethod of claim 25, wherein the disease is colorectal cancer.
 48. Themethod of claim 25, wherein the disease is lung cancer.
 49. The methodof claim 25, wherein the disease is liver cancer.
 50. The method ofclaim 25, wherein the disease is gastric cancer.
 51. The method of claim25, wherein the disease is pancreatic cancer.
 52. The method of claim25, wherein the disease is a hematological malignancy.
 53. The method ofclaim 25, wherein the disease is kidney cancer.
 54. The method of claim25, wherein the disease is skin cancer.
 55. The method of claim 25,wherein the disease is brain cancer.
 56. The method of claim 25, whereinthe disease is prostate cancer.
 57. The method of claim 25, wherein thedisease is head and neck cancer.
 58. The method of claim 25, wherein thedisease is a sarcomas.
 59. The method of claim 25, wherein the diseaseis glioma.